Calreticulin (CRT) and gp96 (GRP94) are endoplasmic reticulum-derived molecular chaperones that elicit potent and effective immune responses that lead to the inhibition and rejection of a variety of tumors and their metastases. The efficacy of chaperones in eliciting immune responses against murine tumors has led to more than a dozen recent and ongoing human clinical trials, including current phase-Ill protocols to evaluate chaperone-induced suppression of human melanoma and renal carcinoma. Therefore, the broad goal of the proposed research is to understand molecular mechanisms of chaperone immunogenicity to enable rational and effective clinical use of these proteins. Antigen-presenting cells (APCs) are required for chaperone-mediated anti-tumor responses, which derive from: 1) stimulation of antigen-independent innate immune responses including APC maturation, activation and cytokine secretion, and 2) activation of the adaptive immune system by eliciting peptide- specific immune responses against associated antigens. At present, the mechanisms by which chaperones elicit these responses are poorly understood. The objective of the proposed studies is to define the mechanisms by which chaperones elicit immune responses from APC. We will do so by investigating: 1) How do chaperones access the APC MHC class-l antigen presentation pathway? 2) How do chaperones function as adjuvants? 3) What is the contribution of Scavenger Receptor Class-A in mediating chaperone-elicited immune responses? We recently identified a novel role for the scavenger receptors SR-A and SREC-1 as endocytic receptors of both gp96 and CRT: expression of Scavenger Receptor Class-A (SR-A) was sufficient to confer chaperone uptake, while SR-AV" macrophages and dendritic cells were impaired in this function. Moreover, SR-A ligands competed for cross-presentation of gp96-associated peptides. On the basis of these findings, we hypothesize that scavenger receptors function in mediating the immune responses stimulated by gp96 and CRT. Thus, we propose to elucidate the mechanisms by which scavenger receptors mediate the immunological effects of chaperones. We will use cellular and molecular techniques to identify the mechanisms by which chaperone complexes elicit antigen-specific responses, and genetic and immunological techniques to evaluate the contribution of scavenger receptors towards chaperone-mediated antigen presentation. Insights obtained will benefit the field of immunotherapy by elucidating the basis of a treatment that is currently undergoing clinical evaluation. These insights will facilitate the rational development and application of this anti-tumor therapy, and the understanding of chaperones as immunological molecules.